This project is centered on determining mechanisms underlying leukocyte interactions with the microvascular wall (endothelial cells, ECs) in vivo. While a great deal of information about relevant local biophysical and biological phenomena is available, relatively little of this has been directly coupled to events occuring in blood perfused microvessels in situ. Our primary focus therefore is on understanding how hydrodynamic phenomena, or specified leukocyte populations, interact with the vessel wall in this "intact" environment. The majority of the work will be undertaken in the blood perfused microvasculature of anesthetized mice. Hypothesis 1A: Adhesion molecule expression is not different among different venule classes, but within individual venules local variation is significant, due to differences in expression among individual endothelial cells. Hypothesis 1B: Non-uniformities in local distribution of adhesion molecules correlate with neutrophiI rolling and adhesive behavior. Distributions of immunofluorescently labeled adhesion molecules will be determined in blood perfused venules using confocal microscopy. We will ask whether heterogeneity in adhesion molecule expression maps to the same population of individual ECs, in both controls and after activation with TNFalpha. Neutrophil rolling and adhesion will be correlated with regions of high immunofluorescence intensity for each adhesion molecule, and with local wall shear rate determined using 0.5mu fluorescent beads to define radial velocity profiles from stacked confocal images. With Project 5, the local hydrodynamic involvement in leukocyte-EC interactions will be explored. Hypothesis 2A: Calcium dependent activation of endothelial cells by inflammatory mediators is necessary for neutrophil adhesion and diapedesis. Hypothesis 2B: Inflammatory mediators increase endothelial cell calcium only in some endothelial cells, and these cells are preferentially associated with neutrophil transmigration. We will use Fluo-4 loaded ECs to determine whether EC Ca 2+ increases occur in individual ECs, or in the venule as a whole, during local exposure to inflammatory mediators, and to relate the responses in EC Ca 2+ to expression of adhesion molecules in individual ECs, and to the hemodynamic environment. We will determine whether leukocytes themselves, independently of the action of inflammatory agents, can initiate Ca 2+ dependent signaling in ECs in venules in their native environment. Neutrophil adhesion density will be spatially and temporally correlated with changes in Fluo-4 intensity, in both control and activated venules, and during buffering of EC Ca2+ changes with BAPTA.